Progress 09/01/23 to 08/31/24
Outputs
Target Audience:We have been directly contacting representatives of whey hydrolysate and other protein hydrolysate manufacturers on both research and development and production sides regarding our results to gauge their interest and better understand how our advances would impact their production processes. We have been seeking feedback to further improve our material, to understand what testing we would need to perform to meet their requirements, and to better simulate production conditions. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?GBS has maintained our relationship with potential customers and has had several email exchanges with their research group to answer questions and provide WPI and WPC used in experiments. They have maintained their interest and GBS expects them to be an early adopter of the ImmobiZyme technology. Business development personnel from GBS attended the IFT First conference in July. While the focus of attendance was not milk protein hydrolysates, GBS utilized the opportunity to promote the technology under development in specific cases. Business Development also talked to a pea protein manufacturing company about opportunities to adapt the technology for production of pea protein hydrolysate. They thought there were opportunities as some enzymes are particularly expensive and reuse would be very attractive. We have been looking for opportunities to present results of our testing at a scientific conference in the coming year to share results with interested researchers and industry representatives. Initial steps towards a website that focused on the immobilized enzyme product, ImmobiZyme™, have been taken. What do you plan to do during the next reporting period to accomplish the goals?Goal 2, Objective 4: As we have completed the evaluation of single cleaning process steps, we are now going to combine them into a recommended cleaning process that produces a 3-log reduction in viable bacteria on pellets, while retaining >95% pellet enzyme activity pre-to-post wash. This process will likely involve rinsing the beads with one cleaning fluid, and then washing them with a second. We will also investigate whether the harsher cleaners can be used intermittently, for example every 5th cleaning, to maximize enzyme lifetime while maintaining the reduction in bacteria on the pellet surface. The final verification will use this established cleaning method for a multi-stage batch test to demonstrate its efficacy. Goal 3, Objective 5:We anticipate that the report from our TABA vendor on the gaps in existing data required for GRAS or other regulatory approval will be submitted to GBS by the end of November. This information will allow us to identify what information is missing for regulatory safety concerns and make plans to address those gaps. Goal 3, Objective 6: At this time, we anticipate that some toxicology testing will be required to support a future regulatory filing for ImmobiZyme. From prior studies of similar materials, one or more tests for genotoxic potential, such as bacterial reverse mutation, in vitro mammalian chromosomal aberration, and in vivo mammalian micronucleus tests; and repeated-dose or acute oral toxicity evaluations using rat models will need to be performed to support regulatory claims of safety. Based on the report returned from our TABA vendor we will seek out accredited vendors to perform various toxicology tests to maximize the information we will get from toxicology testing. Goal 4, Objective 7: Producing ImmobiZyme pellets at the 1-10 kg scale per batch will require adapting the methods and formulations refined during work on Goal 1 of this project. We have already begun determining the appropriate scale equipment to develop a dedicated manufacturing process. Over the course of meeting this objective, we will define systems that: 1) blend the support matrix materials together under the appropriate time and temperature conditions, 2) filter and transfer the matrix materials through a multiport bead formation manifold, 3) complete the pellet formation process, including wash steps to remove fines and unreacted components, 4) complete the enzyme crosslinking process, and 5) finishing and drying the beads. Investigations into different bead formation methods have started to identify the best way to directly transition the bench-scale process to mass production of uniform shaped particles. These pellet formation methods will initially be evaluated at 25-50 g scales to select which method is optimal for pilot-scale production based on uniformity of pellets produced. The pilot-scale process will be incrementally integrated and monitored to define essential points of control along the production pathway. Identifying these critical points such as the rate of pellet addition and the optimum time/temperature for enzyme addition will identify necessary areas and processes for QC monitoring. Additionally, opportunities to recycle waste streams will be identified to reduce waste. The final products of this process will be beads coated with immobilized enzyme, that demonstrate enzyme activity, size uniformity, and physical stability will be within 5% of lab-scale design parameters. Goal 5, Objective 8:The 5th and final goal of this project is to evaluate the pilot scale materials both internally and offer them to external labs for parallel testing. Objective 8 focuses on internal testing at a pilot scale. In this case reactions will be performed in 1-10 L batches. The process parameters will follow optimum reaction conditions identified from work on Goal 1 and utilize the bead cleaning procedures defined in Goal 2. ImmobiZyme bead performance will be evaluated in terms of total substrate conversion, retained pellet activity, and recovered bead mass over a series of consecutive batch runs of 6-24 hours each, depending on reaction temperatures and the substrate. Pellets sampled between batches will be tested for enzyme activity to confirm that any losses are within previously established limits. The reaction substrate conversion will be analyzed and compared to the products of commercial enzyme counterparts. The goal for these beads is to retain more than 99% of their mass between batches and more than 95% of their activity, including losses to any washing between batches. Goal 5, Objective 9: Several of the manufacturers we have had discussions with showed interest in evaluating our ImmobiZyme beads in their laboratories. Following the success of the in-house pilot-scale product evaluations we will reach out to these organizations, and after securing appropriate material transfer agreements, send them kg quantities of the pilot scale material for their evaluation. The test conditions to be used will be selected by these companies to match their standards for new material validation testing. This effort will not only provide outside lab verification of our material performance, but also provide insight into other use conditions and potentially serve to increase interest in our product.
Impacts
What was accomplished under these goals?
Goal 1: The accomplishments related to our first goal focused on optimizing the stability of the immobilized enzyme bead, defining its performance across a broad range of temperatures, and demonstrating the durability of the new bead formulation through 30 consecutive reactions. The initial focus of this goal was on optimizing the bead formation with respect to stability. To this end, different formulations were produced by varying the mass ratios of the primary components of the bead. Reaction conditions were also investigated including crosslinker concentration, reaction time, and temperature. A fractional factorial experimental design was used to reduce the number of necessary tests from several thousand to less than 50, to more efficiently determine the primary effects of each of the parameters. The bead candidates were evaluated for durability by agitating 0.25 g of pellets in an 8000D mixer/mill (Spex SamplePrep) for 1 min., both with and without a 6.35 mm steel ball. After shaking, the beads were separated on a 0.5 mm sieve and the retained beads were weighed. The two most durable formulations were selected for further testing. Objective 1: Objective 1's focus was to demonstrate the beads would retain 99% of their starting mass by the end of the 30-reaction series. For each reaction, a sample of each immobilized enzyme bead was mixed with a 10 wt.% substrate solution for 4 hours. The 30-round bead performance goal was met by the immobilized Alcalase beads, while the immobilized Protemax beads retained 90% of their starting mass. The beads with immobilized lactase surprisingly broke apart much faster, retaining only 50% of their mass after 10 rounds. Follow-on testing identified steps in the immobilization process specifically used for lactase as potentially disrupting the structure of the beads. Further testing using a modified immobilization process is underway to confirm this issue was resolved.? Objective 2: The second objective of this goal was to determine the reactivity of the immobilized enzymes at reaction temperatures between 4-60 oC, and the activity of the pellets following aging at temperatures between 30-80 oC. The immobilized proteases and lactase demonstrated enzyme activity at all tested temperatures. The maximum activity for the immobilized proteases was between 30-40 oC, and the maximum activity for the immobilized lactase was between 50-60 oC. The activity of temperature-aged, immobilized enzyme samples was compared to similar beads that had been stored dry at 4 oC. Both immobilized proteases and the immobilized lactase maintained the same enzyme activity across each tested point up to the final tested time of 168 hours, for all tested temperatures. Goal 2: The two Objectives of Goal 2 focus on development of an immobilized enzyme cleaning process, with Objective 3 focusing on a cleaning method that retains enzymatic activity and Objective 4 focusing on removal of bacteria from the immobilized enzyme beads. Model microbes for these tests were selected from common dairy contaminants: E. faecium, B. cereus, B. licheniformis, B. subtilis, G. stearothermophilus, and S. thermophiles. Cleaning solutions for the beads were selected from CIP solutions that are commonly used in food and dairy processing plants: tap water, bleach, ppm peracetic acid (PAA), and a representative surfactant (Tergitol L-61, Dow). Testing towards this goal is roughly 50% complete. The concentration and time of exposure for PAA and bleach solutions have been identified for effective cleaning of the beads while retaining enzyme activity. An elevated temperature tap water rinse also demonstrated reasonable efficacy in removing bacteria while not inhibiting the enzymatic activity of the beads. A challenge we have come across in this testing is that some of the bacteria naturally do not adhere well to the beads. This makes achieving the goal of 3-log removal of bacteria difficult. Staring with lower concentrations of bacteria on the control samples, lead to results from the removal approach that are at or below the limit of detection of bacterial measurement protocol. Goal 3:Efforts towards this goal have focused mainly on Objective 4, identification of the gaps in existing data required for GRAS or other regulatory approval. We met with our TABA vendor in July . We have had several email exchanges since then. The vendor has been progressing with their determination on the need for GRAS. Their target is to have a report to GBS by the end of November 2024. Efforts towards Objective 5 will progress from this report. Goal 4: Initial steps have been taken in identifying components that will successfully scale-up the lab production process. A primary focus has been on the components that produce the initial pellet/bead forms. Goal 5:Work towards Goal 5 will start following the completion of the first production batches from Goal 4.
Publications
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